Patent NZ-630856-A (NZ)
Title: Mitochondrial toxicity test
Inventor: SJÖVALL FREDRIK
IPC: G01N33/50
Filed: 2013-10-04
Abstract: An in vitro method for screening and selection of potential drug candidates or for testing a person’s sensitivity to a substance with effect on mitochondria is disclosed. The method comprises i) subjecting a sample of human blood cells containing mitochondria obtained previously from a person to high-resolution respirometry; ii) adding to the sample a substance that increases the leakage of the inner mitochondrial membrane to protons, thereby activating electron transport and increasing oxygen consumption of the mitochondria; iii) adding to the same sample, a test drug substance in a vehicle and iv) comparing the effect on oxygen consumption before and after addition of the test drug substance with the effect on oxygen consumption of vehicle only. A relative decrease in oxygen consumption of the test drug substance indicates a negative effect on the mitochondria, and a relative increase in oxygen consumption of the test drug substance indicates a positive effect on mitochondrial electron transport. The method further comprises v) adding to the sample from iii) an inhibitor of mitochondrial complex I-function; and vi) adding to the sample from v) an inhibitor of mitochondrial complex III-function. The inhibitor of mitochondrial complex I-function and complex III-function are added sequentially to provide a residual, non-mitochondrial respiration to enable evaluation of complex-specific effects on mitochondrial respiration and reveal non-specific properties of the test drug substance. An in vitro method for investigating mitochondrial effects of drug candidates in clinical trials or in treatment regimens is disclosed where the method comprises i) subjecting a sample of human blood cells containing mitochondria obtained previously from a person to high-resolution respirometry. The sample of cells is from a person subjected to a clinical study or to a treatment regimen, and a test drug substance has been administered to the person during the clinical study or treatment regimen. A substance that increases the leakage of the inner mitochondrial membrane to protons is added to the sample, thereby activating electron transport and increasing oxygen consumption of the mitochondria and then the oxygen consumption of the sample from the person subjected to the clinical study or to a treatment regimen is compared with the oxygen consumption of a control sample. A lower oxygen consumption rate of the sample from the person subjected to the clinical study or to a treatment regimen indicates a negative effect on the mitochondrial function, and a higher oxygen consumption rate of the sample from the person subjected to the clinical study or to a treatment regimen indicates a positive effect on mitochondrial electron transport. An inhibitor of mitochondrial complex I-function is added to the sample and then an inhibitor of mitochondrial complex III-function is added to the sample. The Inhibitor of mitochondrial complex I-function and complex III-function are added sequentially to provide a residual, non-mitochondrial respiration to enable evaluation of complex-specific effects on mitochondrial respiration and reveal non-specific properties of the test drug substance that was administered to the person subject to the clinical study or to a treatment regimen. Disclosed is an in vitro method for screening and selecting potential drug candidates or for testing the sensitivity of a person to a substance with effect on mitochondria comprising: i) subjecting a sample of intact human blood containing mitochondria obtained previously from a subject to high-resolution respirometry; ii) adding to the sample a substance that inhibits complex I respiration; iii) adding to the sample a drug test substance in a vehicle, wherein an increase in oxygen consumption of the test drug substance indicates membrane permeability and a positive effect on mitochondrial electron transport; iv) adding to the sample a substance that permeabilizes the plasma membrane. A lack of increase in oxygen consumption following permeabilization in the presence of the test drug substance indicates membrane permeability, and an increase in oxygen consumption indicates limited membrane permeability of the test drug substance. The method further comprises v) adding to the sample a reference substance, wherein the reference substance is a non-membrane permeable complex II-linked substrate, where the effect of the said reference substance on oxygen consumption enables evaluation of the function of complex II and potential toxicity of the test drug substance; and vi) adding to the sample a substance that inhibits complex III respiration to reveal and compensate for non-specific properties of test drug substances.